JPH08131157A - Maltose phosphorylase, trehalose phosphorylase and new strain belonging to genus plesiomanas capable of producing these enzymes - Google Patents

Abstract

PURPOSE: To obtain the subject new bacterium belonging to the genus Plesiomonas, capable of producing maltose phosphorylase and trehalose phosphorylase enzymes required for producing trehalose useful for medicines, cosmetics and foods. CONSTITUTION: This new bacterium Plesiomonas SH-35 (FERM P-5,144) belonging to the genus Plesiomonas, capable of producing maltose phosphorylase and trehalose phosphorylase, is isolated from sludge of Tagonoura seashore at FuJi City in the Shizuoka Prefecture in Japan. The new strain is cultured in the presence of maltose as a carbon source, an enzyme is formed/accumulated and collected to give maltose phosphorylase which reversibly phosphorylates maltose to form glucose and β-D-glucose phosphate and trehalose phosphorylase which reversibly phosphorylates trehalose to form glucose and β-D-glucose 1-phosphate. Trehalose is obtained from maltose by the reversible reaction of these enzymes.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism, a novel enzyme and a method for producing a novel enzyme. More specifically, a novel microorganism belonging to the genus Plesiomonas having the ability to produce maltose phosphorylase and trehalose phosphorylase necessary for enzymatically producing trehalose, a novel maltose phosphorylase and trehalose phosphorylase obtained from this microorganism, and The present invention relates to a method for producing these enzymes.

[0002]

BACKGROUND OF THE INVENTION Trehalose is a substance expected to have a wide range of applications in medicine, cosmetics, foods and the like. Therefore, many attempts have been made to industrially produce trehalose. These technologies can be roughly classified into three types. One of them is a method of extracting and purifying the substance from a microorganism having a property of accumulating trehalose in cells [Journal of American Chemical Society (J. Am. Chem. Soc.) Volume 72,
2059, 1950, German Patent No. 266584, JP-A-3-130084, JP-A-5-91890, JP-A-5-184353, JP-A-5-292986]. This method comprises a step of culturing a microorganism, a step of separating the microorganism, a step of extracting trehalose from the microorganism, and a step of purifying and crystallizing the extracted trehalose, and the manufacturing step is very complicated. Moreover, not only the productivity of trehalose is lower than that of other methods, but also a large amount of microbial extraction residue is generated as waste, so it cannot be said to be an economically efficient method.

As another method, microorganisms that produce trehalose extracellularly (in the medium) are searched, and the genus Brevibacterium and Corynebacterium (C) are searched.
A fermentation method has been developed in which a microorganism such as orynebacterium) is cultured to produce trehalose extracellularly (in the medium) [Japanese Patent Laid-Open No. 5-218882]. However, even with this method, the accumulation rate of trehalose in the medium was about 3%.
Since the yield is as low as (w / v), a large-capacity fermenter and a refining facility commensurate with it are required to mass-produce trehalose on an industrial scale, which is economically problematic.
Moreover, in the present method as well, in order to obtain purified trehalose, not only a sterilization operation is required, but also a complicated step is required for removal of contaminants other than trehalose produced by the strain used during culture or medium components, etc. I am trying.

On the other hand, an enzymatic method has already been developed as a method for solving various problems of these fermentation methods at once. This includes maltose phosphorylase (Malto
se: orthophosphate β-D-glucosyltransferase) and algae-derived trehalose phosphorylase (α, α-Trehalos
e: orthophosphate β-D-glucosyltransferase) is allowed to act on maltose in the presence of phosphoric acid to produce trehalose (Patent No. 1513517, Agri.Biol.Chem., 49).
Vol., 2113, 1985), and bacterial sucrose phosphorylase (Sucrose: orthophosphate α-D-glucose).
syltransferase) and basidiomycete-derived trehalose phosphorylase (α, α-Trehalosep: orthophosphate α-D-glucose
syltransferase) on sucrose in the presence of phosphate to obtain trehalose (Abstracts of the 6th Annual Meeting of the Japan Society for Agricultural Chemistry, 1994, 3Ra14).

According to these methods, it has been reported that trehalose is produced from maltose or sucrose in a high yield of 60 to 70%. Further, in this method, since the raw material used is a purified high-purity saccharide, it is easy to purify trehalose obtained by an enzymatic reaction, and it is considered to be an industrially advantageous method compared to other methods. Has been. However, the enzymes also used in these methods,
In particular, the source of trehalose phosphorylase is algae or basidiomycetes such as Euglena and Maitake, and producing enzymes from these has not only economic problems but also technical difficulties. Moreover, not only is trehalose phosphorylase obtained and sucrose phosphorylase and maltose phosphorylase used in combination with it very difficult to control the pH when used in combination because the optimum pH range of each enzyme is greatly different. , Very low temperature stability,
The trehalose formation reaction could be performed only at a low temperature of about 25 to 37 ° C. This suggests that contamination of various bacteria occurs during the enzymatic reaction carried out using an open-type reaction tank, and strict sanitary control is required to prevent secondary reactions due to this. have. Furthermore, when these known enzymes are used in combination, a high-concentration raw material could not be used due to the substrate concentration dependence of these enzymes. Therefore, this method was not economically efficient.

[0006] From the above, if a new maltose phosphorylase and trehalose phosphorylase that are easy to produce and purify, have high thermostability, and are independent of substrate concentration can be found, maltose can be obtained easily and in large quantities. It is possible to easily and efficiently produce trehalose with high yield as a raw material.

[0007] Therefore, a first object of the present invention is to provide a novel microorganism capable of producing the novel enzymes maltose phosphorylase and trehalose phosphorylase satisfying the above various conditions with high production efficiency. A second object of the present invention is to provide a new maltose phosphorylase and trehalose phosphorylase which are easy to produce and purify, have high thermostability, and are independent of substrate concentration. A third object of the present invention is to provide a method for producing the above-mentioned two types of enzymes simply and in high yield using the above-mentioned microorganism.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have sought to obtain a microorganism capable of producing an enzyme having these various properties that maltose phosphorylase and trehalose phosphorylase must have for industrial use. Widely searched the natural world. As a result, they have found that a microorganism belonging to the genus Plesiomonas produces a large amount of both enzymes having the above requirements, and completed the present invention.

[0009] That is, the present invention relates to a microorganism belonging to the genus Plesiomonas, which has the ability to produce maltose phosphorylase and trehalose phosphorylase, and Life Science Article No. 5144.

The present invention further relates to maltose phosphorylase having the following physicochemical properties. (A) Action: The α-1,4-glucopyranoside bond in maltose is reversibly phosphorolytically decomposed in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): Acts on maltose,
Does not act on other disaccharides. (C) Optimum pH and stable pH range: optimum p of decomposition reaction
H is 7.0 to 7.5, and the optimum pH of the synthetic reaction is 6.
0. Under heating conditions of 50 ° C. and 10 minutes, the pH is 5.
It is stable within the range of 5 to 7.0. (D) Temperature stability: It is stable up to 45 ° C under heating conditions of pH 6.0 and 15 minutes. (E) Optimum action temperature range: The optimum action temperature for the decomposition reaction is around 50 ° C., and the optimum action temperature for the synthetic reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating condition of 50 ° C. for 10 minutes
Complete inactivation at H5.0 and 8.0. Also, the pH
It is completely deactivated at 55 ° C. in the treatment for 6.0 minutes. (G) Inhibition: copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercuribenzoate,
Inhibited by sodium dodecylbenzene sulfonate. (H) Isoelectric point by isoelectrofocusing method: 3.8 (ri) Molecular weight by SDS polyacrylamide gel electrophoresis: about 92,000 (Molecular weight by gel filtration method is about 20)
10,000, and is composed of two subunits).

The present invention further relates to trehalose phosphorylase having the following physicochemical properties. (A) Action: The α-1,1-glucopyranoside bond in trehalose is reversibly phosphorolytically decomposed in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): It acts on trehalose and does not act on other disaccharides. (C) Optimum pH and stable pH range: The optimum pH for the decomposition and synthesis reactions is 7.0. Under heating conditions of 50 ° C. and 10 minutes, the pH is in the range of 6.0 to 9.0. (D) Temperature stability: It is stable up to 50 ° C. under heating conditions of pH 7.0 and 15 minutes. (E) Range of suitable action temperature: The suitable action temperature for the decomposition reaction and the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating condition of 50 ° C. for 10 minutes
Complete inactivation at H5.0 and 9.5. Also, the pH
It is completely deactivated at 55 ° C. in the treatment for 7.0 minutes. (G) Inhibition: Inhibited by copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercuribenzoate. (H) Isoelectric point by isoelectrofocusing method: 4.5 (i) Molecular weight by SDS polyacrylamide gel electrophoresis: about 88,000 (Molecular weight by gel filtration method is about 20)
10,000, and is composed of two subunits).

In addition, the present invention provides the above-mentioned Plesiomonas (Ple
culturing a microorganism of the present invention belonging to the genus siomonas), producing and accumulating at least one of the maltose phosphorylase of the present invention and the trehalose phosphorylase of the present invention, and collecting the maltose phosphorylase and trehalose phosphorylase. Manufacturing method. The present invention will be described below.

The novel strain of the present invention has been newly isolated by the present inventors from sludge on the coast of Tagagoura in Fuji City, Shizuoka Prefecture. This strain is based on the Burgers Manual
Of Determinative Bacteology (Bergey's
Mannual of Determinative Bacteriolgy), 8th Edition, Volume 1, the mycological properties of the isolated strains are shown in Table 1 below, as shown in Table 1 below.
seiomonas) and was identified as a related fungus of P. shigelloides. However, the contents described in that the VP test and the urease test are positive, that D-mannose, D-galactose, L-arabinose, and D-fructose can be assimilated, and that they can grow even under alkaline conditions of pH 9 In addition, it is significantly different from known strains in that both trehalose phosphorylase and maltose phosphorylase are produced and accumulated in the cells and in the medium (outside the cells). The strain Plesiomonas SH-35 has been deposited at the Institute of Biotechnology, National Institute of Industrial Science and Technology as No. 5144 (FERM BP-5144).

[0014]

[Table 1]

The new strain of the present invention was screened as follows. First, the collected coastal sludge was suspended in a saline solution, and 1 drop of the suspension was spread on an agar medium having the following composition.
The agar plate medium used was agar 2% (w / v), trehalose or maltose 1%, polypeptone 0.5%, yeast extract 0.5%, dipotassium phosphate 0.1%, magnesium sulfate / heptahydrate. Contains 02%. Thus, the agar plate was cultivated aerobically at 37 ° C. to obtain each colony appearing on the plate, and each colony was cultured in a liquid medium having the above composition for 24 to 72 hours at 37 ° C. in the liquid medium having the above composition. 180r.
p. m. The culture was performed with shaking. Then, add each culture to 12,0
The cells were centrifuged at 00 × g for 10 minutes at 4 ° C. to separate the cells and the supernatant. The bacterial cells thus obtained contain a small amount of 0.1M.
The cells were suspended in a phosphate buffer (7.0) and the activity was measured by the method described below. As a result, a strain having the above-mentioned mycological characteristics could be isolated.

The novel microorganism of the present invention is a bacterium that produces novel maltose phosphorylase and trehalose phosphorylase. Next, a method for producing these enzymes will be described. The microorganism of the present invention (FERM BP-5144) is inoculated into an appropriate medium and cultured. From the viewpoint of the growth temperature of the cells, it is suitable to culture the cells in a temperature range of 25 to 42 ° C. for 8 to 70 hours under aerobic conditions. By culturing the microorganism of the present invention, the trehalose phosphorylase and / or maltose phosphorylase of the present invention is produced. Most of the produced enzyme is accumulated inside the microbial cells, and a part is accumulated outside the microbial cells (in the medium). Therefore, the maltose phosphorylase and / or trehalose phosphorylase produced and accumulated inside or outside the cells (in the medium) are collected. Further, the above culture can be carried out by either a batch system or a continuous system.

The medium used for the above culture will be described.
As the carbon source, trehalose, maltose and sugars containing them can be used. Various organic and inorganic nitrogen compounds may be used as the nitrogen source, and the medium may further contain various inorganic salts. When trehalose or a sugar containing the substance is used as a carbon source,
The microorganism of the present invention preferentially produces trehalose phosphorylase. When maltose or a sugar containing the substance is used as a carbon source, the microorganism of the present invention simultaneously produces maltose phosphorylase and trehalose phosphorylase. However, in this case, the amount of trehalose phosphorylase produced tends to be smaller than that when trehalose is used as the carbon source. Furthermore, by using both trehalose and maltose as a carbon source or a sugar containing these substances, trehalose phosphorylase and maltose phosphorylase can be simultaneously produced, and by controlling the amount of trehalose and maltose, trehalose phosphorylase. It is also possible to control the production rate of maltose phosphorylase.

The nitrogen source is corn steep liquor,
Inorganic nitrogen sources such as soybean meal or various peptones, and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium phosphate, urea and the like, which are inexpensive compounds generally used for culturing microorganisms, can be used. Needless to say, the urea and organic nitrogen sources also serve as carbon sources.

A medium suitable for use in the method of the present invention is, for example, trehalose 1-2% when preferentially producing trehalose phosphorylase.
(W / v), yeast extract 2%, ammonium phosphate 0.1
5%, urea 0.15%, common salt 1%, dipotassium phosphate 0.
It is appropriate to use a liquid medium having a pH of 7.0 to 7.5 containing 1%, magnesium sulfate / heptahydrate 0.02% and calcium carbonate 0.2%. Moreover, when it is desired to simultaneously produce maltose phosphorylase and trehalose phosphorylase, maltose 1-2% (w / v), polypeptone S (manufactured by Nippon Pharmaceutical Co., Ltd.) 2-3%, ammonium phosphate 0.15%, urea 0.15% It is suitable to use a liquid medium containing 1% sodium chloride, 0.1% dipotassium phosphate, 0.02% magnesium sulfate heptahydrate and 0.2% calcium carbonate.

As mentioned above, when maltose is used as the carbon source, not only maltose phosphorylase but also
Trehalose phosphorylase is also produced in a certain amount. Therefore, in order to obtain a crude enzyme for trehalose production (mixture of maltose phosphorylase and trehalose phosphorylase), it is convenient and economical to use maltose as a carbon source.

Each enzyme accumulated in the cultured cells or in the culture supernatant can be isolated by a conventional method. First, the enzyme in the bacterial cell can be used as a crude enzyme together with the bacterial cell.
Further, the crude enzyme can be recovered by extracting the enzyme from the bacterial cells. Further, the enzyme is also contained in the culture supernatant outside the cells, and the remaining culture solution from which the cells have been separated can be used as the crude enzyme-containing solution. Further, these crude enzymes can be purified by a usual method such as a solvent precipitation method using ethanol, acetone, isopropanol and the like, ammonium sulfate fractionation method, ion exchange chromatography, gel filtration chromatography and the like. Furthermore, the separation of maltose phosphorylase and trehalose phosphorylase can be performed by anion exchange chromatography because the isoelectric points of the two are different.

The enzyme of the present invention thus obtained is
As described in detail in Examples described later, the maltose phosphorylase and trehalose phosphorylase having the above-mentioned physicochemical properties. Since the Plesiomonas SH-35 strain of the present invention does not produce α-glucosidase (maltase) that hydrolyzes maltose or trehalose, glucoamylase, trehalase, etc., the activity of these enzymes can be determined by measuring the phosphorolysis reaction. A simple method is applicable in which glucose is produced by enzymatic reaction in the presence of phosphate with maltose or trehalose as the substrate, and the glucose is produced by the glucose oxidase method. The synthetic reaction can be performed by using a mixed solution of β-D-glucose monophosphate and glucose as a substrate and measuring the inorganic phosphate produced by the enzymatic reaction by a conventional method.

Enzyme activity measuring method (1) Decomposition reaction: 0.01 ml of enzyme solution was added to 0.5 ml of 20 mM maltose or trehalose solution dissolved in 50 mM phosphate buffer solution (pH 7) and reacted at 50 ° C. for 15 minutes. After that, heat in a boiling water bath for 3 minutes to stop the enzyme reaction. Then, after cooling in running water, the produced glucose is measured by the glucose oxidase method (Wako Pure Chemical Industries, Ltd., glucose C-II Test Wako). Here, 1 unit of enzyme activity is 1 per minute under the same conditions.
It is defined as the amount of enzyme that produces μmole (micromole) of glucose. (2) Synthetic reaction: 0.05 ml in 0.15 ml of a mixed solution of 27 mM β-D-glucose monophosphate Na salt dissolved in 70 mM HEPES buffer (pH 7.0) and glucose at the same concentration. Add the enzyme solution of 50 ℃
After reacting for 15 minutes, heat in a boiling water bath for 2 minutes to stop the enzyme activity. Then, the produced inorganic phosphoric acid is measured using Wako Pure Chemical Industries, Ltd. P-test Wako. Here, 1 unit of enzyme activity is the amount of enzyme that produces 1 micromol (μmole) of inorganic phosphoric acid per minute under the same conditions.

The novel enzymes maltose phosphorylase and trehalose phosphorylase of the present invention can be used as a crude enzyme or a purified enzyme, respectively. Furthermore, it is also possible to use, for example, trehalose, microbial cells having the activity of both enzymes and immobilized microbial cells in which the microbial cells are entrapped, adsorbed or chemically bound to a suitable carrier. Furthermore, the enzyme of the present invention can be used as an immobilized enzyme in which each enzyme is immobilized by a known method.

[0025]

EXAMPLES The present invention will be further described below with reference to examples. Example 1 Production of intracellular and extracellular maltose phosphorylase and
Purified Plesiomonas SH-35 strain (FERM BP-5144), maltose 1% (w / v), Polypeptone S (Nippon Pharmaceutical Co., Ltd.) 2
%, Ammonium phosphate 0.15%, urea 0.15%, common salt 1%, dipotassium phosphate 0.1%, magnesium sulfate.
The cells were inoculated in a liquid medium of pH 7.0 containing 0.02% of heptahydrate and 0.2% of calcium carbonate. Next, this liquid medium was aerobically cultured at 37 ° C. for 24 hours. The obtained culture broth was centrifuged at 12,000 xg for 15 minutes at 4 ° C to separate the cells and the supernatant. The obtained cells are a small amount of 20 mM
After suspending in a phosphate buffer (pH 7.0), the cells were disrupted by an ultrasonic cell disruptor. Then, ammonium sulfate was added to the disrupted cell suspension to make it 30% saturated, and the mixture was allowed to stand at 4 ° C. overnight. Then, the mixture was centrifuged to remove the precipitate, and ammonium sulphate was further added to the obtained supernatant to make it 70% saturated. Precipitates formed by standing overnight at 4 ° C. were collected by centrifugation, dissolved in 20 mM phosphate buffer (pH 7), and then dialyzed thoroughly against the same buffer.

Then, the solution was passed through a DEAE-Fractogel (manufactured by Merck) column equilibrated with the same buffer to adsorb the enzyme. The adsorbed enzyme was eluted with a concentration gradient method of 0 M to 0.6 M sodium chloride contained in the same buffer, and then concentrated with a UF membrane (Amicon, YM-30). The concentrated enzyme was purified by gel filtration chromatography using a Sephacryl S-300 (Pharmacia) column equilibrated with the same buffer containing 0.2 M sodium chloride. The obtained active fractions were collected, dialyzed thoroughly with the same buffer containing 1.5 M ammonium sulfate, and then passed through a phenyl-Toyopearl (Tosoh) column equilibrated with the same buffer containing 1.5 M ammonium sulfate. Then, the enzyme was adsorbed. The adsorbed enzyme is contained in the same buffer solution as above.
After elution with a concentration gradient method of 5 M to 0 M ammonium sulfate, the obtained active fractions were collected and sufficiently dialyzed against the same buffer containing 0.2 M sodium chloride. After concentration using the above-mentioned UF membrane, 0.
Gel filtration chromatography was performed again on Superdex 200 (Pharmacia) equilibrated with the same buffer containing 2M sodium chloride, and the obtained active fraction was concentrated by the above-mentioned method and polyacrylamide gel disk electrophoresis ( P
AGE) and SDS-PAGE method, uniform intracellular maltose phosphorylase purified enzyme (activity yield of about 28
%)was gotten. The extracellular enzyme was also purified and concentrated in the same manner as above using the culture supernatant as a starting material to obtain a purified enzyme of maltose phosphorylase with an activity yield of about 25%.

Example 2 Production of intracellular and extracellular trehalose phosphorylase
And purified Plesiomonas SH-35 strain (FERM BP-5144) with trehalose 1% (w / v), yeast extract 2%, ammonium phosphate 0.15%, urea 0.15%, salt 1%, dipotassium phosphate. 0.1%, magnesium sulfate, heptahydrate 0.02%
And a liquid medium of pH 7.0 containing 0.2% of calcium carbonate. This liquid medium was cultured in the same manner as in Example 1 and post-treated to obtain a disrupted cell suspension and a supernatant. The obtained disrupted cell and supernatant fluids were purified in the same manner as in Example 1. Homogeneous intracellular trehalose phosphorylase and extracellular trehalose phosphorylase were obtained in the PAGE method and the SDS-PAGE method at activity yields of 35% and 32%, respectively.

Example 3 Maltosephos produced by Plesiomonas SH-35 strain
Enzymaticization of holylase and trehalose phosphorylase
Various Properties Next, the Plesiomonas SH-35 strain (FERM BP-51 of the present invention
Regarding the general enzymatic chemical properties of the novel maltose phosphorylase and trehalose phosphorylase produced by 44), the results obtained by using the purified enzyme obtained by purifying by the same method as in Examples 1 and 2 are shown below. As a result of the preliminary experiment, both the intracellular enzyme and the extracellular enzyme showed almost the same physicochemical properties, and therefore the various properties of the intracellular enzyme are shown here.

(A) Action 1% dissolved in 10 mM phosphate buffer (pH 7.0)
To the (w / v) maltose and trehalose solution, 5 units (decomposition reaction) of maltose phosphorylase and trehalose phosphorylase, respectively, were added to 1 g of the substrate, reacted at 50 ° C. for 5 hours, and then heated in a boiling water bath for 3 minutes. As a result of measuring the sugar in the saccharified solution obtained by deactivating the enzyme by high performance liquid chromatography, glucose and glucose monophosphate were respectively detected. 10m
Mixed solution of 1% (w / v) glucose and β-D-glucose monophosphate Na salt or α-D-glucose monophosphate Na salt dissolved in M Tris hydrochloric acid buffer (pH 7.0) As a substrate, and 5 units of maltose phosphorylase and trehalose phosphorylase were added to 1 g of the substrate and reacted at 50 ° C. for 5 hours, and then treated as described above to measure the sugar composition. As a result, glucose and β-D- Maltose and trehalose were detected from glucose 1-phosphate, but a disaccharide synthesis reaction from glucose and α-D-glucose 1-phosphate was not detected.

The produced sugar was analyzed by the following method. That is, the insoluble matter in the saccharified solution obtained by heating and deactivating is 0.4
The filtrate obtained by filtering with a 5 μm membrane filter was used as a test sugar solution, and YMC-Pack, ODS-AQ (A
(Q-304, YMC) column was used for the measurement. Water was used as the mobile phase, the column temperature was 30 ° C., and a differential refractometer was used for detection. (B) Substrate specificity (decomposition reaction) The activity when various saccharides were used as the substrate in place of the substrate (maltose or trehalose) shown in the above-mentioned activity measurement method (decomposition reaction) was expressed as relative activity. Table 2 shows the results
Shown in

[0031]

[Table 2]

(C) Optimum pH and stable pH range The optimum pH of the decomposition and synthesis reactions was measured using purified enzyme. As a result, as shown in A of FIG. 1, the optimum pH of the decomposition reaction of maltose phosphorylase (shown by a white circle) is 7.0 to 7.5, and the synthesis reaction (shown by a black circle) is 6.
0 was optimal. Further, as shown in FIG. 1B, trehalose phosphorylase had an optimum decomposition reaction (white circle) and synthetic reaction (black circle) of 7.0. In the case of pH decomposition reaction, a 20 mM phosphate buffer solution was used, and in the case of synthesis reaction, MES (pH 5.5 to 6.5) and MOPS (pH
Value 6.5-7.0), HEPES (pH 7.0-8.
0) and Tris-hydrochloric acid (pH 7.5 to 9.0). In addition, as a result of treating both of the purified enzymes in each buffer solution at 50 ° C. for 10 minutes and measuring their residual enzyme activities by a decomposition reaction, as shown in FIG. 2, maltose phosphorylase (white circles) had a pH of 5. In the range of 5 to 6.5, trehalose phosphorylase (black circle) is p
It was stable from H 6.0 to 9.0. The pH is acetic acid (pH 5.0 to 5.5), phosphoric acid (pH 6.0 to 8.
It adjusted using each buffer solution of 0) and carbonic acid (pH 8.9).

(D) Optimal temperature of action As a result of measuring the optimal temperature of the decomposition reaction of both enzymes and the synthetic reaction, as shown in FIG. 3, maltose phosphorylase (A, white circle: decomposition reaction, black circle: synthetic reaction), trehalose phosphorylase. (B, white circle: decomposition reaction, black circle: synthetic reaction) In both cases, the decomposition reaction was in the range of 50 ° C and the synthesis reaction was in the range of 50 to 55 ° C. (E) Temperature-dependent inactivation conditions Maltose phosphorylase and trehalose phosphorylase are treated for 15 minutes under the conditions of pH 6.0 and 7.0, which are stable pHs, respectively, and the remaining inactivation is compared with the untreated state by a conventional method. It was measured by. As a result, as shown in FIG. 4, the maltose phosphorylase (open circle) was 55.
At ℃, trehalose phosphorylase (black circle) is 6
It was completely deactivated at 0 ° C. (F) Inhibitor The activity of both enzymes in the synthesis reaction was measured in the presence of various inhibitors, and the results are shown in Table 3 as relative activity with 100% activity without addition.

[0034]

[Table 3]

[0035] The isolation electro focusing method using (g) Isoelectric point Aisogeru (FMC BioProducto Inc.), a result of measuring the isoelectric point of both enzymes, maltose phosphorylase, as shown in FIG. 5 is 3.8, trehalose The phosphorylase was 4.5. (H) Molecular weight The molecular weight of both purified enzymes was measured by the SDS-PAGE method and the gel filtration method using Sephacryl S-200.
As a result, as shown in FIG. 6, the molecular weight of both enzymes was about 200,000 by the gel filtration method, but SDS-PA was used.
Maltose phosphorylase was about 92,000 in the GE method.
Since trehalose phosphorylase was about 88,000, it was expected that each of these enzymes was composed of two subunits. The above-mentioned physicochemical properties are compared with those of known maltose phosphorylase and trehalose phosphorylase.
Summarized in.

[0036]

[Table 4]

[0037]

[Table 5]

Example 4 Production of intracellular and extracellular trehalose phosphorylase
Method Trehalose 1% (w / v), yeast extract (Difco)
2%, ammonium phosphate 0.15%, urea 0.15%,
It was obtained by culturing overnight in the same medium as above in 20 liters of a liquid medium of pH 7.5 containing sodium chloride 1%, dipotassium phosphate 0.1%, magnesium sulfate / heptahydrate 0.02% and calcium carbonate 0.2%. Plesiomonas SH-35 strain inoculum (FE
1 liter of RM BP-5144) is aseptically added, and the temperature is 37 ° C for 3
00 rpm, ventilation amount 1 v. v. The culture was performed with aeration and stirring under the condition of m for 24 hours. As a result of measuring the trehalose phosphorylase activity of the main culture solution, it was 1.5 units per 1 ml of the culture solution. Similarly, the maltose phosphorylase activity was also measured, but the activity was very small. Then 12,0
Centrifugation at 00 × g for 10 minutes at 4 ° C. gave about 160 g of bacterial cells (when wet) and 19.5 liter of supernatant, which was concentrated with Romicon UF membrane (YM-30). ,
About 1 liter (about 6,400 units) of extracellular concentrated crude enzyme was obtained. As a result of measuring the enzyme activity in the supernatant, about 25% (7.5 × 10 ³ units) of the total activity (about 30 × 10 ³ units) was measured.
Units) of activity. Moreover, about the bacterial cell part, 10
Thoroughly wash with mM phosphate buffer (pH 7), 500 ml
After the cells were suspended in the same buffer as above, the cells were disrupted with an ultrasonic cell disrupter, and the trehalose phosphorylase activity was measured by a conventional method. As a result, about 75% of the total activity (22.5 × 10 ³
The unit) was contained in the cells.

Example 5 (Production of cells containing maltose phosphorylase and trehalose phosphorylase and extracellular enzyme) Trehalose in the medium components used in Example 1 was maltose, and yeast extract was Polypeptone FC (manufactured by Nippon Pharmaceutical Co., Ltd.). ) 5%
(SH / SH), instead of SH-3
Five strains (FERM BP-5144) were cultured. As a result of measuring the maltose phosphorylase activity and the trehalose phosphorylase activity of the main culture liquid, 0.5 unit of maltose phosphorylase activity and 0.45 unit of trehalose phosphorylase were contained per 1 ml of the culture liquid. Then, similarly centrifuge to obtain about 150 g of bacterial cells (when wet) and 1
9.6 liters of supernatant was obtained. Then, the results of measuring the maltose phosphorylase activity in the cells and the supernatant,
About 78% (about 1%) of the total activity of maltose phosphorylase
20,000 units were contained inside the cells, and about 22% were contained outside the cells (culture supernatant). In addition, about 82% (about 9,000 units) of the total activity of trehalose phosphorylase was contained in the cells and about 18% was contained outside the cells (culture supernatant). The culture supernatant was concentrated in the same manner as in Example 1,
About 1 liter of concentrated enzyme was obtained, which contained about 1,700 units of maltose phosphorylase and 1,43 units.
It contained 0 units of trehalose phosphorylase.

[0040]

INDUSTRIAL APPLICABILITY The present invention provides a novel microorganism capable of producing maltose phosphorylase and trehalose phosphorylase necessary for enzymatically producing trehalose inside and outside the cells (in the medium). Since the microorganism of the present invention is a bacterium, not only the method for obtaining the enzyme is easy as compared with green algae and basidiomycetes, which are conventionally known as a source of trehalose phosphorylase, but the culture time can be significantly shortened, which is economical. Target. Furthermore, the microorganism of the present invention has the advantage that one bacterium can simultaneously produce two enzymes required for the enzymatic production of trehalose. Further, since the two enzymes of the present invention satisfy all the requirements required for enzymatically producing trehalose, the effective use of the obtained trehalose is remarkably facilitated, and a significant improvement in economic terms is achieved. It can be guaranteed. That is, since the maltose phosphorylase and trehalose phosphorylase of the present invention have high temperature stability and can perform an enzymatic reaction at high temperature, contamination with bacteria during the reaction is avoided. Furthermore, since both enzymes have almost the same optimum pH range, there is an advantage that complicated pH control during the reaction is unnecessary.

[Brief description of drawings]

FIG. 1 shows optimum reaction curves of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain [symbol: decomposition reaction (◯), synthetic reaction (●)].

FIG. 2 shows the pH stability of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain [symbol: maltose phosphorylase (◯), trehalose phosphorylase (●)].

[Fig. 3] Optimal action temperature of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain [symbol: decomposition reaction (○), synthetic reaction (●)].

FIG. 4 shows the temperature stability of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain [symbols: maltose phosphorylase (◯), trehalose phosphorylase (●)].

FIG. 5 shows isoelectric points of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain.

FIG. 6: Molecular weight of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas SH-35 strain

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1:01)

Claims (9)

[Claims] 1. A method for producing maltose phosphorylase and trehalose phosphorylase, wherein Plesiomonas (P
lesiomonas) microorganism belonging to the genus Life Science Article No. 5144
issue. 2. A maltose phosphorylase having the following physicochemical properties. (A) Action: The α-1,4-glucopyranoside bond in maltose is reversibly phosphorolytically decomposed in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): Acts on maltose,
Does not act on other disaccharides. (C) Optimum pH and stable pH range: optimum p of decomposition reaction
H is 7.0 to 7.5, and the optimum pH of the synthetic reaction is 6.
0. Under heating conditions of 50 ° C. and 10 minutes, the pH is 5.
It is stable within the range of 5 to 7.0. (D) Temperature stability: It is stable up to 45 ° C under heating conditions of pH 6.0 and 15 minutes. (E) Optimum action temperature range: The optimum action temperature for the decomposition reaction is around 50 ° C., and the optimum action temperature for the synthetic reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating condition of 50 ° C. for 10 minutes
Complete inactivation at H5.0 and 8.0. Also, the pH
It is completely deactivated at 55 ° C. in the treatment for 6.0 minutes. (G) Inhibition: copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercuribenzoate,
Inhibited by sodium dodecylbenzene sulfonate. (H) Isoelectric point by isoelectrofocusing method: 3.8 (ri) Molecular weight by SDS polyacrylamide gel electrophoresis: about 92,000 (Molecular weight by gel filtration method is about 20)
10,000, and is composed of two subunits). 3. A trehalose phosphorylase having the following physicochemical properties. (A) Action: The α-1,1-glucopyranoside bond in trehalose is reversibly phosphorolytically decomposed in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): It acts on trehalose and does not act on other disaccharides. (C) Optimum pH and stable pH range: The optimum pH for the decomposition and synthesis reactions is 7.0. Under heating conditions of 50 ° C. and 10 minutes, the pH is in the range of 6.0 to 9.0. (D) Temperature stability: It is stable up to 50 ° C. under heating conditions of pH 7.0 and 15 minutes. (E) Optimum temperature range for action: The optimum temperature range for the decomposition reaction and the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating condition of 50 ° C. for 10 minutes
Complete inactivation at H5.0 and 9.5. Also, the pH
It is completely deactivated at 55 ° C. in the treatment for 7.0 minutes. (G) Inhibition: Inhibited by copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercuribenzoate. (H) Isoelectric point by isoelectrofocusing method: 4.5 (i) Molecular weight by SDS polyacrylamide gel electrophoresis: about 88,000 (Molecular weight by gel filtration method is about 20)
10,000, and is composed of two subunits). 4. Maltose characterized by culturing the microorganism according to claim 1, producing and accumulating at least one of the maltose phosphorylase according to claim 2 and the trehalose phosphorylase according to claim 3, and collecting the maltose. Method for producing phosphorylase and trehalose phosphorylase. 5. The method according to claim 4, wherein the culture is carried out in the presence of maltose as a carbon source to produce and accumulate maltose phosphorylase and trehalose phosphorylase. 6. The production method according to claim 4, wherein the culture is performed in the presence of trehalose as a carbon source to preferentially produce and accumulate trehalose phosphorylase. 7. A microbial cell is separated from the culture broth after culturing, and the separated bacterial cell is directly used as maltose phosphorylase and / or
Alternatively, the production method according to claim 4, which is a crude enzyme of trehalose phosphorylase. 8. The method according to claim 4, wherein cells are separated from the culture solution after the culture, and a crude enzyme of maltose phosphorylase and / or trehalose phosphorylase is extracted from the separated cells. 9. The method according to claim 4, wherein the cells are separated from the culture solution after the culture, and the obtained culture supernatant solution is used as a crude enzyme-containing solution of maltose phosphorylase and / or trehalose phosphorylase.